JPH0622795A - Method for improving performance of chemical emission analysis using peroxidase as labeling substance - Google Patents

Abstract

PURPOSE:To perform the chemical emission analysis in high sensitivity while preventing a non-specific reaction, by reacting a peroxidase, luminol and H2O2 in the presence of a radical stabilizer, adding skim milk, albumen albumin, etc., to the reaction solution, and measuring the amount of the emission. CONSTITUTION:The chemical emission analysis of an analysis target substance is performed by making a specific binding reagent such as an antibody capable of specifically binding to the analysis target substance bind with a peroxidase for labeling the reagent, reacting the labeled reagent with a specimen, reacting the peroxide of the labeled product of the reagent bound to the analysis target substance in the specimen with luminol and hydroperoxide in the presence of a radical stabilizer {e.g. 4-[4'-(2'-methyl)thiazolyl]phenol}, adding one kind or more of skim milk, albumen albumin, protein-decomposed product, a saccharide alcohol, a non-ionic surfactant, etc., to the reaction solution, and subsequently detecting and measuring the amount of the emission in the reaction solution with a photoelectric tube, etc. Thereby, a non-specific emission reaction, namely a reagent blank, is lowered, and/or a specific emission reaction is increased to enhance the reliability of the measurement value.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for improving the performance of chemiluminescence analysis used for peroxidase as a labeling substance, and is useful for the analysis and quantification of trace amounts of biological components in the field of clinical examination.

[0002]

2. Description of the Related Art As a method for sensitively detecting and measuring a substance to be analyzed, a specific binding reagent such as an antibody that specifically binds to the substance (often labeled with an enzyme such as peroxidase in advance) There is known a method (so-called enzyme immunoassay method, etc.) of reacting a sample with a sample and subsequently detecting and measuring a signal generated by a catalytic reaction of an enzyme such as peroxidase which is a labeled substance. As the enzyme immunoassay, various improved methods or modified methods such as a one-antibody method, a two-antibody method, a sandwich method, a homogeneous method, and a heterogeneous method are known.

As a method of efficiently measuring the signal generated by the catalytic reaction of an enzyme such as peroxidase, there is a method of measuring the chemiluminescence of luminol / hydrogen peroxide catalyzed by peroxidase. Radical stabilizers that aid electron oxidation are used as sensitizers (Methods in Enzymology, Vol.133, p.
331-353, 1986; JP-A-2-291299).

On the other hand, it is also known that the activity of peroxidase is increased by adding polyoxyethylene ethers (Clinica Chimica Acta, 109 , 177-181, 198).
1; J. Clin. Chem. Clin. Biochem., 19 , 435-439, 198
1), and a method for increasing the S / N ratio or the measurement sensitivity by including polyoxyethylene ethers in a buffer solution in the enzyme immunoassay method is also disclosed (Tokuheihei 2-503).
No. 029 publication).

Further, conventionally, proteins such as albumin have been often immobilized on a solid phase carrier for the purpose of blocking non-specific reaction of antibodies.

[0006]

When the substance to be analyzed is an ultratrace substance in the living body, etc., development of a method having higher sensitivity than the above method is desired. The present invention aims to develop a method that is even more sensitive than conventional methods.

[0007]

Means for Solving the Problems In the chemiluminescence analysis using peroxidase in the presence of a radical stabilizer (sensitizer), the present inventors have conducted a nonspecific luminescence reaction, that is, a reagent blank (hereinafter referred to as noise). ), And / or a specific luminescence reaction (hereinafter, also referred to as a signal).
When various methods for enhancing the S / N ratio (signal / noise ratio) were investigated, skim milk powder, ovalbumin, protein, etc. were added to the reaction solution when detecting and measuring the amount of luminescence in the reaction solution. It was found that the addition of one or more of a decomposed product, a sugar alcohol and a nonionic surfactant achieves the above object, and completed the present invention.

That is, the present invention provides the following (1) to (8):
Regarding (1) In the chemiluminescence analysis in which the peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer, and the generated luminescence amount is detected and measured, the luminescence amount in the reaction solution is measured. A method for improving the performance of chemiluminescence analysis by allowing one or more of skim milk powder, ovalbumin, proteolytic products, sugar alcohols and nonionic surfactants to be present in the reaction solution during detection / measurement. (2) In a chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence generated, the amount of luminescence in the reaction solution is measured. A method for improving the performance of chemiluminescent immunoassay, in which skim milk powder is present in the reaction solution during detection and measurement. (3) In a chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced, the amount of luminescence in the reaction solution is measured. A method for improving the performance of chemiluminescence analysis by allowing ovalbumin to be present in the reaction solution during detection and measurement. (4) In a chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the generated luminescence amount, the luminescence amount in the reaction solution is A method for improving the performance of chemiluminescence analysis, in which a proteolytic product is present in the reaction solution during detection and measurement. (5) The above (1), wherein the protein degradation product is casein degradation product.
Or the method of (4). (6) In a chemiluminescence analysis in which a labeled peroxidase is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced, the amount of luminescence in the reaction solution is measured. A method for improving the performance of chemiluminescence analysis by allowing sugar alcohol to be present in the reaction solution during detection and measurement. (7) The method according to (1) or (6) above, wherein the sugar alcohol is mannitol or sorbitol. (8) In a chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced, the amount of luminescence in the reaction solution is measured. A method for improving the performance of chemiluminescence analysis, in which a nonionic surfactant is present in the reaction solution during detection and measurement.

The chemiluminescence analysis in the present invention is particularly preferable as long as it is an analysis method in which peroxidase as a labeling substance is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer, and the generated luminescence is detected and measured. The present invention is not limited, and can be used for various analysis methods such as a one-antibody immunoassay method, a two-antibody immunoassay method, a competitive analysis method, a sandwich method, a homogeneous method, a heterogeneous method, a Western analysis method, and a DNA probe method.

The peroxidase used in the present invention is
Horseradish basic isozymes are preferred. The horseradish basic isozymes are known to be of the B, C, D and E types, of which the C type is most preferred.

As the luminol used in the luminescence reaction, hydrazine and sulfide ions, which are the starting materials for production, are often mixed in the commonly available reagent grade luminol. Therefore, purified luminol is used after purification.

As the radical stabilizer used in the present invention, phenol derivatives such as p-iodophenol and 4- [4 '-(2'-methyl) thiazolyl] phenol, 6
There are benzothiazole derivatives such as hydroxybenzothiazole, 3- (10-phenothiazyl) -propyl sulfonate, and the like.

When the luminol / hydrogen peroxide luminescent reaction is carried out by the catalytic action of peroxidase in the presence of these radical stabilizers, the radical stabilizer becomes a sensitizer radical, and this sensitizer radical becomes luminol. Reacts and emits light.

The higher the pH of the reaction, the stronger the amount of luminescence, but the luminescence that does not depend on the catalytic activity of peroxidase increases. Therefore, considering both factors, it is advisable to carry out the reaction within the range of 7.0 to 11.0.

The skim milk powder used in the present invention may be a commercially available product. The concentration used is such that at least the S / N ratio is improved. Usually, the concentration is 0.
It is in the range of 01 to 0.5% (weight / volume%, hereinafter the same), preferably 0.02 to 0.2%. The concentration is 0.01
%, The effect of improving the S / N ratio is small and 0.5
If it exceeds%, the specific luminescence reaction is suppressed, which is not preferable.

Commercially available ovalbumin can be used in the present invention. The concentration used is such that at least the S / N ratio is improved. Usually, the concentration is 0.01 to 0.5%, preferably 0.02 to 0.2% in the final concentration of the reaction solution.
Is the range. If the concentration is less than 0.01%, the effect of improving the S / N ratio is small, and if it exceeds 0.5%, the specific luminescent reaction is suppressed, which is not preferable.

The protein degradation products used in the present invention include enzymatic degradation products and acid degradation products of proteins such as casein and soybean protein. The concentration used is such that at least the S / N ratio is improved. Usually, the concentration is in the range of 0.01 to 0.5%, preferably 0.02 to 0.2%, as the final concentration of the reaction solution. If the concentration is less than 0.01%, the effect of improving the S / N ratio is small, and if it exceeds 0.5%, the specific luminescent reaction is suppressed, which is not preferable.

Examples of the sugar alcohol used in the present invention include 6 to 4 monosaccharide alcohols such as mannitol, sorbitol, galactitol, ribitol, arabitol and erythritol. The concentration is such that at least the S / N ratio is improved. Usually, the concentration is in the range of 0.01 to 10%, preferably 0.25% to 2.0% as the final concentration of the reaction solution. If the concentration is less than 0.01%, the effect of improving the S / N ratio is small, and if it exceeds 10%, the specific luminescence reaction is suppressed, which is not preferable.

As the nonionic surfactant used in the present invention, there are acyl sorbitan, polyoxyethylene octyl phenyl ethers, octyl phenol ethylene oxide, polyoxyethylene sorbitan esters and the like, which are Atlas Powder C.
o.) Manufacturing span 20 (sorbitan monolaurate),
Span 40 (Sorbitan Monopalmitate), Span 6
0 (sorbitan monooleate), span 80 (sorbitan monostearate), span 85 (sorbitan trioleate), tween 20 (polyoxyethylene sorbitan monolaurate), tween 40 (polyoxyethylene sorbitan monopalmitate), tween 60
(Polyoxyethylene sorbitan monostearate),
Tween 80 (polyoxyethylene sorbitan monooleate), Tween 85 (polyoxyethylene sorbitan trioleate), Bridge 35 (polyoxyethylene lauryl ether) and Bridge 58 (polyoxyethylene cetyl ether), Rohm and Haas Company (Rohm & Haas Co.) manufactured Triton X-100 (polyoxyethylene (10) octyl phenyl ether) and Triton X-405 (polyoxyethylene (40) octyl phenyl ether), or nonidet P-40 (octyl phenol) from Sigma. Nonionic surfactants sold under the trade name of ethylene oxide) are available.

The concentration of the nonionic surfactant added is such that at least the S / N ratio is improved. Normal,
The final concentration of the reaction solution is 0.001 to 2.0%,
More preferably, it is in the range of 0.01 to 0.5%. If the concentration is less than 0.001% or more than 2%, the effect of improving the S / N ratio is small.

Among the above additives, skim milk powder is characterized by the effect of significantly reducing the amount of noise. Ovalbumin has almost no effect on the signal amount, but is characterized by the effect of reducing the amount of noise, maintaining the generated luminescence stably, and further enhancing the storage stability of the luminescent substrate solution. The proteolytic product has almost no effect on the signal amount, but has the effect of reducing the noise amount.
Sugar alcohol has almost no effect on the signal amount, but has the effect of reducing the noise amount. The nonionic surfactant has almost no effect on the noise amount, but is characterized by the effect of increasing the signal amount.

[0022]

【Example】

Example 1 Skim milk powder on reagent blank (noise),
Effect of ovalbumin, protein degradation product, sugar alcohol, or nonionic surfactant The test without additives (control) was carried out as follows.
That is, a 50 mM sodium borate buffer solution (pH 10.0; hereinafter referred to as a borate buffer solution for light emission) is applied to a strip type microwell having an aluminum reflective film deposited on the outer surface.
100 μl, 2 mM 4- [4 ′-(2′-methyl) thiazolyl] phenol dimethyl sulfoxide solution 8 μm
l, 4 mM hydrogen peroxide / light emitting borate buffer 50 μl and 20 mM luminol solution (manufactured by Hitachi Chemical Co., Ltd.) 50 μl
Take l, stir and mix, chemiluminescence measuring device (Corona Electric
The chemiluminescence amount was measured for 20 minutes with the use of MLR-100 manufactured by Co., Ltd.

On the other hand, in the additive addition test, skim milk powder, ovalbumin, casein degradation product, sugar alcohol, or nonionic surfactant was used at the final concentration of the reaction solution instead of 100 μl of the borate buffer solution for luminescence. The same operation as above was performed using 100 μl of the borate buffer solution for luminescence contained at the concentration shown in Table 1. As a result, the reagent blank was almost constant during the reaction (20 minutes). Further, the reagent blank (noise) at the reaction time of 10 minutes was as shown in Table 1.

[0024]

[Table 1] Table 1 Effect of nonfat dry milk, ovalbumin, protein hydrolyzate, sugar alcohol, or nonionic surfactant on reagent blank ━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━ Additive final concentration Reagent blank ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━% Lumicount-free (control) -240 Skim milk powder 0.05 50 〃 0.10 30 Egg albumin 0.05 120 〃 0.10 100 Casein degradation product 0.05 140 〃 0.10 200 Mannitol 0 .25 120 0.45 65 sorbitol 0.45 65 Tween 20 0.05 233 Tween 80 0.05 236 Triton X-100 0.05 234 Nonidet P-40 0.05 239 Bridge 35 0.05 38 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

From the results shown in Table 1, skim milk powder significantly reduces the reagent blank, and ovalbumin, casein hydrolyzate and sugar alcohol reduce the reagent blank to a moderate level, while nonionic surfactants such as Tween 20 It can be seen that it has almost no effect on the reagent blank.

Example 2 Effect of Sugar Alcohol on Reagent Blank Value (Noise) in the Presence of Nonfat Dry Milk and Tween 20 0.1% nonfat dry milk and 0% were added to a strip type microwell having an aluminum reflective film deposited on the outer surface. 2% Tween 20
100 μl of borate buffer for luminescence containing 2 mM 4-
8 μl of a solution of [4 ′-(2′-methyl) thiazolyl] phenol in dimethylsulfoxide, 4 mM containing mannitol or sorbitol in the concentrations shown in Table 2 below.
Hydrogen peroxide / Borate buffer for light emission 50 μl and 20 m
50 μl of M luminol solution was taken, and the same procedure as in Example 1 was performed, and the chemiluminescence amount was measured. As a control, 4 mM hydrogen peroxide containing no sugar alcohol / 50 μl of borate buffer for light emission was used instead of 4 mM hydrogen peroxide containing 50 μl of borate buffer for light emission. It was operated and measured (Table 2).

[0027]

[Table 2] Table 2 Effect of sugar alcohol on reagent blank in the presence of skim milk powder and Tween 20 ━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━ Sugar alcohol final concentration Reagent blank ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━% Lumicount No Addition (control) 0.0 30 Mannitol 0.25 15 〃 0.45 8 Sorbitol 0.45 8 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━

From the results of Table 2, skim milk powder and Tween 2
It can be seen that in the presence of 0, both mannitol and sorbitol further reduce the reagent blank.

Example 3 Effect of skimmed milk powder, ovalbumin or protein degradation product on reagent blank in the presence of mannitol 0 parts of skim milk powder, egg white albumin or protein degradation product were placed in strip type microwells having an aluminum reflective film deposited on the outer surface. 100 μ of borate buffer for luminescence containing .10% (final concentration: 0.05%) or 0.2% (final concentration: 0.1%)
1, 2 mM 4- [4 ′-(2′-methyl) thiazolyl] phenol in dimethylsulfoxide solution 8 μl, 1.0
% Mannitol-containing 4 mM hydrogen peroxide / luminescent borate buffer 50 μl and 20 mM luminol solution (Hitachi Chemical
50 μl (manufactured by Co., Ltd.) was taken, and the chemiluminescence amount was measured by the same procedure as in Example 1. As a control, skim milk powder,
In place of 100 μl of the borate buffer for luminescence containing ovalbumin or a protein degradation product, 100 μl of the borate buffer for luminescence not containing these was used, and the measurement was performed in the same manner as above (Table 3).

[0030]

[Table 3] Table 2 Effect of skim milk powder, albumen albumin, and proteolytic products on reagent blank in the presence of mannitol ━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━ Protein or final concentration Reagent blank Protein degradation product ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ -% Lumicount (control) 0.0 70 Nonfat dry milk 0.05 20 〃 0.10 10 Ovalbumin 0.05 30 〃 0.10 24 Casein hydrolyzate 0.05 35 〃 0.10 54 ━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

From the results in Table 3, it is understood that skim milk powder, ovalbumin and casein degradation products all reduce the reagent blank in the presence of mannitol. Of these, skim milk powder reduced the reagent blank most significantly.

Example 4 Effect of mannitol on signal amount The signal amount based on a specific reaction was determined as follows in the case where no additive was added (control). That is, horseradish peroxidase (TOYOBO Grade I-
C) Labeled anti-endothelin-1 monoclonal antibody (Yamasa Shoyu Co., Ltd., MCA ET-02) diluted 100,000 times (diluent: borate buffer for light emission) 100 μl, 2 mM 4
-[4 '-(2'-Methyl) thiazolyl] phenol dimethyl sulfoxide solution 8 µl, 4 mM hydrogen peroxide / light emitting borate buffer solution 50 µl and 20 mM luminol solution 50 µl were taken and mixed with stirring, and a chemiluminescence measuring device was used. The amount of luminescence was measured immediately after mixing, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes after mixing, respectively. On the other hand, in the case of adding mannitol, 4 mM hydrogen peroxide containing mannitol (final concentration: 0.25%) instead of 4 mM hydrogen peroxide / 50 μl of borate buffer for light emission /
The same procedure as above was performed using a borate buffer for luminescence (Table 4).

From the results shown in Table 4, it can be seen that the signal amount is slightly stronger when mannitol is added for 5 minutes after the start of photometry, as compared with when no addition (control) is performed. There was no significant difference between the two when the time exceeded 5 minutes after the start of photometry.

[0034]

[Table 4] Table 4 Effect of mannitol on the signal level ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ) Signal- free (control) 0.25% mannitol ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Lumi Count Lumi Count 0 1382 1495 1 1461 1544 2 1480 1553 3 1488 1546 4 1489 1529 5 1485 1511 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━

Example 5 Effect of Nonionic Surfactant on Reagent Blank, Signal Amount or S / N Ratio in the Presence of Mannitol In a strip type microwell having an aluminum reflective film deposited on the outer surface, horseradish peroxidase was added. A 100,000-fold diluted solution of the labeled anti-endothelin-1 monoclonal antibody (diluent: a luminescent borate buffer solution containing each nonionic surfactant shown in Table 5 at a final concentration of 0.02 to 1.0%). Use) 100 μl, 2 mM 4- [4 ′-(2′-methyl) thiazolyl] phenol in dimethylsulfoxide 8 μl, 4 mM containing 1.0% mannitol
Hydrogen peroxide / Luminescent borate buffer 50 μl and 20 mM
50 μl of a luminol solution was sequentially added, mixed with stirring, and the chemiluminescence amount was measured for 20 minutes using a chemiluminescence measuring device.

As a control, 100 μl of the boric acid buffer for luminescence containing each nonionic surfactant was replaced with 100 μl of the borate buffer for luminescence not containing the same, and the same operation as above was performed. did. FIG. 1 shows the time course of the signal amount with and without each nonionic surfactant (0.05% at the final concentration) (control). The reagent blank remained almost constant during the reaction time of 20 minutes.
In addition, Table 5 shows the reagent blank (noise), the signal amount, and the S / N ratio at the reaction time of 10 minutes.

[0037]

[Table 5] Table 5 Effect of non-ionic surfactant on reagent blank, signal amount or S / N ratio in the presence of mannitol ━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━ Surfactant final concentration Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━% Lumicount No Lumicount (control) 278 450 450 1.62 Tween 20 0.02 238 1311 5.51 0.05 05 302 13634 .51 0.10 334 1303 3.90 Tween 80 0.02 303 1293 4.27 0.05 05 402 1354 3.37 0.10 362 1257 3.47 Triton X-100 0.02 366 1331 3.64 0. 05 390 1357 3.48 0.10 70 1233 4.57 Nonidet P40 0.02 339 1197 3.53 0.05 0.05 340 1195 3.51 0.10 404 1283 3.18 Bridge 35 0.02 290 1058 3.65 0.05 310 310 1155 3.730 .10 348 1229 3.53 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

From the results shown in FIG. 1 and Table 5, the tween 20,
Tween 80, Triton X-100, Nonidet P-4
0 or the one to which the bridge 35 was added increased the signal amount by 4 to 5 times (although hardly affecting the reagent blank) as compared with the one to which these were not added (control), and S / N Similarly, it can be seen that the ratio is also higher than that of the non-addition (control).

Example 6 Effect of Coexistence of Skim Milk Powder and Nonionic Surfactant on Reagent Blank, Signal Amount or S / N Ratio in the Presence of Mannitol In a strip type microwell having an aluminum reflective film deposited on the outer surface, A 100,000-fold diluted solution of anti-endothelin-1 monoclonal antibody labeled with horseradish peroxidase (diluting solution: 0.05% final concentration skim milk powder and 0.05% final concentration)
A borate buffer for luminescence containing 10% of each nonionic surfactant was used. ) 100 μl, 2 mM 4- [4'-
(2'-Methyl) thiazolyl] phenol dimethyl sulfoxide solution 8 μl, 4 mM hydrogen peroxide containing 1.0% mannitol / light emitting borate buffer 50 μl
50 μl of a 20 mM luminol solution were mixed with stirring, and the chemiluminescence amount was measured for 20 minutes using a chemiluminescence measuring device.

As a control, instead of the skim milk powder and each nonionic surfactant-containing luminescent borate buffer solution, a luminescent borate buffer solution containing no nonionic surfactant was used, and the same procedure as above was performed. It was measured. FIG. 2 shows the time-dependent change in signal amount. The reagent blank remained almost constant during the reaction time of 20 minutes. In addition, Table 6 shows the reagent blank (noise), the signal amount, and the S / N ratio at the reaction time of 10 minutes.

From the results of Table 6, skim milk powder and Tween 2
The combination of 0, Tween 80 or Triton X-100 increased the amount of luminescence 3 to 7 times as compared with the non-addition (control), and the S / N ratio was also combined with the noise reduction effect of skim milk powder. It can be seen that the concentration is increased to about 20 times that of no addition (control).

[0042]

[Table 6] Table 6 Effect of coexistence of skimmed milk powder and nonionic surfactant on reagent blank, signal amount or S / N ratio ━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━ Skim milk powder or surfactant Noise (N) Signal (S) S / N ratio ━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Lumicount No Lumicount (control) 254 467 1.84 Skim milk powder + Tween 20 27 1075 39.81 Skim milk powder + Tween 80 27 994 36.81 nonfat dry milk + Triton X-100 27 1179 43.67 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━

Example 7 Effect of Coexistence of Ovalbumin and Nonionic Surfactant on Stable Persistence of Signal Level in the Presence of Mannitol Black microfluorolimovawell (manufactured by Dynatech) was treated with horseradish peroxidase. A 100,000-fold diluted solution of the labeled anti-endothelin-1 monoclonal antibody (dilution: a borate buffer for luminescence containing 0.05% final concentration ovalbumin and 0.05% final concentration Tween 20 was used). 100 μl, 2 mM 4- [4 '-(2'
-Methyl) thiazolyl] phenol in 8 μl in dimethylsulfoxide, containing 1.0% mannitol 4
mM hydrogen peroxide / light emitting borate buffer 50 μl and 2
Take 50 μl of 0 mM Luminol solution, stir and mix,
The amount of chemiluminescence was measured over time for 20 minutes using a chemiluminescence measuring device. For comparison, the same operation was performed except that skim milk powder of the same concentration (final concentration 0.05%) was used instead of ovalbumin. FIG. 3 shows the change in signal amount over time. It was found that the addition of ovalbumin has a more stable and sustained signal amount than that of skim milk powder.

Example 8 Storage stability of peroxychemical hydrogen reagent containing ovalbumin or skim milk powder The storage stability of a hydrogen peroxide reagent containing ovalbumin and a hydrogen peroxide reagent containing skim milk powder was compared. The hydrogen peroxide reagent containing ovalbumin is 1% mannitol, 0.
15% ovalbumin, 0.1% Tween 20 and 4 m
It was prepared by appropriately diluting it with 50 mM sodium borate buffer (pH 10.0) so as to contain M hydrogen peroxide. Also, the hydrogen peroxide reagent contained in skim milk powder should contain 1% mannitol, 0.075% skim milk powder, 0.1% Tween 20 and 4 mM hydrogen peroxide, respectively.
It was prepared by appropriately diluting with 0 mM sodium borate buffer (pH 10.0). Both of the above hydrogen peroxide reagents were stored at 37 ° C, and a certain amount of them was sampled over time,
Using this, a chemiluminescence reaction was performed and the amount of luminescence was measured.

The chemiluminescence reaction was carried out by adding anti-endothelin-1 monoclonal antibody labeled with horseradish peroxidase to 0.2%.
50m containing 075% ovalbumin (Seikagaku Corporation)
Dilute 1 million times with M sodium borate buffer (pH 10.0), take 100 μl of this, and then add 2 mM
4- [4 '-(2'-methyl) thiazolyl] phenol dimethylsulfoxide solution 8 μl and the above hydrogen peroxide test solution 5
0 μl and 20 mM luminol solution (Hitachi Chemical Co., Ltd.)
(Manufactured by Co., Ltd.), 50 μl in this order, was injected into a black microfluororemover well (manufactured by Dynatech), stirred, and photometrically measured using a chemiluminescence measuring device (Corona Denki Co., Ltd., MLR-100). The amount of luminescence after 9 minutes was taken as the measured value (Fig. 4). Figure 4
Therefore, ovalbumin is more effective than non-fat dry milk for the storage stability of the hydrogen peroxide reagent.

[0046]

INDUSTRIAL APPLICABILITY The present invention is a method for improving the performance of chemiluminescence immunoassay using peroxidase in the presence of a radical stabilizer, which reduces nonspecific luminescence reaction, that is, reagent blank (noise), And / or enhances the specific luminescent response or amount of signal and thus the S / N
It improves the ratio (signal / noise ratio), sustains and stabilizes luminescence, or enhances storage stability of reagents, and contributes to improvement in reliability of measured values.

[Brief description of drawings]

FIG. 1 is a graph showing a change over time in a reagent blank and a signal amount when various nonionic surfactants coexisted (0.05% at a final concentration) in the presence of mannitol.

[Fig. 2] Both skimmed milk powder and a nonionic surfactant coexist in the presence of mannitol (both 0.05% at the final concentration).
%) Is a graph showing the changes over time in the reagent blank and the signal amount when the concentration was changed.

FIG. 3 is a graph showing a change over time in signal amount when ovalbumin or skim milk powder coexist (0.05% at a final concentration) in the presence of mannitol and a nonionic surfactant.

FIG. 4 is a graph showing storage stability of a peracid chemical hydrogen reagent solution containing ovalbumin or skim milk powder.

[Explanation of symbols]

 1 and 2, □ ─ □: Tween 20 ■ ─ ■: Tween 80 △ ─ △: Triton X-100 ▲ ─ ▲: Nonidet P-40 ◇ ─ ◇: Bridge 35 ● ─ ●: No addition (control) In Fig. 3, □ ─ □: ovalbumin ● ─ ●: skim milk powder In Fig. 4, ◆ ─ ◆: ovalbumin ◇ ─ ◇: skim milk powder

Claims (8)

[Claims] 1. A chemiluminescence assay for reacting a peroxidase, which is a labeling substance, with luminol and hydrogen peroxide, in the presence of a radical stabilizer to detect and measure the amount of luminescence produced. Improving the chemiluminescence analysis performance by allowing one or more of skim milk powder, ovalbumin, proteolytic products, sugar alcohols and nonionic surfactants to be present in the reaction solution when detecting and measuring the amount Method. 2. Luminescence in a reaction solution in chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced. A method for improving the performance of chemiluminescence immunoassay in which skim milk powder is present in the reaction solution when detecting and measuring the amount. 3. Luminescence in a reaction solution in chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced. A method for improving the performance of chemiluminescence analysis, in which ovalbumin is present in the reaction solution when detecting and measuring the amount. 4. Luminescence in a reaction solution in chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced. A method for improving the performance of chemiluminescence analysis, in which a proteolytic product is present in the reaction solution when detecting and measuring the amount. 5. The method according to claim 1 or 4, wherein the protein degradation product is a casein degradation product. 6. Luminescence in a reaction solution in chemiluminescence analysis in which peroxidase, which is a label, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced. A method for improving the performance of chemiluminescence analysis by allowing sugar alcohol to be present in the reaction solution when detecting and measuring the amount. 7. The method according to claim 1 or 6, wherein the sugar alcohol is mannitol or sorbitol. 8. Chemiluminescence analysis in which peroxidase, which is a labeling substance, is reacted with luminol and hydrogen peroxide in the presence of a radical stabilizer to detect and measure the amount of luminescence produced, and the luminescence in the reaction solution is detected. A method for improving the performance of chemiluminescence analysis in which a nonionic surfactant is present in the reaction solution when detecting and measuring the amount.